Research in Progress: This project is in collaboration with: Martin W. Brechbiel Radiation Oncology Branch, National Cancer Institute, National Institutes of Health. James Sellers, Neil Billington and Yasuharu Takagi, Laboratory of molecular physiology, National Heart, Lung, and Blood Institute, National Institutes of Health. and Gopalakrishnan Balasubramanian Max Planck Institute for Biophysical Chemistry Currently, there are two main projects: The first project involves the use of single-molecule techniques to measure the optical properties and characteristics of imaging probes. Because of the complex nature of nano particles used for molecular imaging, it has proved difficult to reliably determine the average number of incorporated fluorophores and the fraction of particles that are labeled using traditional ensemble measurement techniques. In collaboration with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, we used the custom built prism-based total internal reflection fluorescence (TIRF) microscope and single-molecule imaging capabilities in our lab to measure the fluorescence properties of synthesized particles. By measuring the fluorescence from single particles as a function of time, we are able to directly observe the photo-bleaching of individual dyes in the particles. The number of photo-bleaching steps that reduce the fluorescence to background levels is indicative of the number of dyes in each particle. The magnitude of each discrete decrease in intensity is indicative of the brightness of the individual dyes, whereas the time between photo-bleaching steps directly provides the photo-bleaching rate, or the photo-stability of the dye. The wide-field single-molecule TIRF set-up allows the collection of thousands of individual fluorescence traces, providing excellent statistical samples. In a proof-of-principle experiment, we determined the average number of dyes per particle for 15 nm iron core silica particles embedded with either Alexa 555 or Cy 5.5 dyes. Analysis of the fluorescence traces revealed that encapsulation of the dye increased its fluorescence intensity and increased its photo-stability as evidenced by brighter emission and longer bleaching times as compared with free dye. From the distribution of the number of dyes per particle we could infer the fraction of particles that were labeled, which is difficult to ascertain by ensemble methods. We anticipate that this relatively simple, robust and rapid technique that requires trivial amounts of material will be of general interest to the nanoparticle and molecular imaging fields. We are extending this technique to quantify the stoichiometry and labeling efficiency of fluorescently tagged chemotherapeutic antibodies. The long term goal of this research is the establishment of tools, techniques and methodologies to accurately and efficiently characterize the properties of nanomaterials employed in bio medical applications, which is an established unmet need in this field. In a second project we are collaborating with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health on functionalizing and characterizing nitrogen vacancy fluorescent nanodiamonds (FNDs) for use as multi-modal imaging probes. These are attractive fluorescence particless for in vivo and in vitro tracking and imaging studies as they are bright, non-blinking fluorophores that are excited in the green (532 nm) and emit in the far red spectrum (600-700 nm), which has superior tissue penetration and signal-to-noise characteristics compared with shorter wavelengths in biological samples. Moreover, diamond is inert and the fluorescence arises from the nitrogen vacancy so the core particle contains no organic dyes or other potentially toxic material that would be problematic for in vivo applications. Remarkably, the FNDs can be as small as 5 nm, which is also advantageous for biocompatibility and clearing. We have developed a coating and functionalization process that stabilizes nm sized FNDs in solution and provides a facile functionalization scheme that allows them to be specifically attached to bio-molecules. These FND probes enable high-resolution, high speed three dimensional single-molecule tracking over indefinite periods and we anticipate that they will be widely adopted as single-molecule tracking probes. In a third project in collaboration with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, we have developed a new phase-sensitive background-free imaging technique based FNDs. The fluorescence emission of FNDS shows a weak magnetic-field strength dependence. We exploit this phenomenon to selectively modulate the emission of FNDs and use phase sensitive techniques to extract the diamond fluorescence emission from background fluorescence and noise. Fianlly, in collaboration with Jim Sellers, Neil Billington, and Yasuharu Takagi in the Laboratory of Molecular Physiology in the National Heart, Lung, and Blood Institute, we are testing the use of FNDs in optical trapping experiments.